CN103276046A - Method for counting subject matters to be counted in container and device for counting - Google Patents
Method for counting subject matters to be counted in container and device for counting Download PDFInfo
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- CN103276046A CN103276046A CN2013102205853A CN201310220585A CN103276046A CN 103276046 A CN103276046 A CN 103276046A CN 2013102205853 A CN2013102205853 A CN 2013102205853A CN 201310220585 A CN201310220585 A CN 201310220585A CN 103276046 A CN103276046 A CN 103276046A
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Abstract
The present invention provides a method for counting subject matters to be counted in the liquid of a sealed container, wherein at least one part of the thickness of the container is adjusted, at least one part of the range to be adjusted serves as a counting subject range, and the number of the subjects to be counted in the counting subject range is counted. The present invention also provides a device for counting which is used for counting the number of subjects to be counted in the liquid of a sealed container and is characterized by being equipped with a loading platform for loading the container and an adjusting component for adjusting at least one part including the counting subject range to the specified thickness.
Description
201080011026.6), denomination of invention is the division of the international application of " method of counting of the counting object cell culture processes, cell culture apparatus, container in and count and use device " the application's international application no that to be the applicant propose on March 2nd, 2010 is PCT/JP2010/001414 (China national phase application number:.
Technical field
The present invention relates to method of counting, counting device for the counting object in cell culture processes, cell culture apparatus and the container of the cells of culturing cell, tissue, microorganism etc.
Background technology
In recent years, in fields such as the production of pharmaceuticals, gene therapy, regenerative medicine, immunotherapy, require under artificial environment efficiently, culturing cell, tissue, microorganism etc. in large quantities.
In a large amount of cultivations of this cell, particularly when cultivating suspension cell, it is comparatively general using the stir culture of the culture tank that possesses agitating wing usually.But, particularly form under the situation of cohesion piece limit proliferating cells at the cell that is subjected to the external force damage easily or limit, widespread use is not used agitating wing and cell is enclosed in the culture vessel, leave standstill (cell sinks under the state of bottom surface), cultivate, along with the propagation of cell, transfer in the bigger container of floorage or increase the method for container number.But, leave standstill in the cultivation at this, exist when the cell condensation piece increases along with the propagation of cell, the oxygen of each cell and the supply of nutritive ingredient are become not enough, the problem that proliferate efficiency descends gradually.
In addition, when transfer vessel, though can first stir culture liquid and the inequality of oxygen and nutritive ingredient is eliminated,, the damage that when existing because of each jump operation cell is produced makes the problem of proliferate efficiency decline.
On the other hand, also extensively carry out the shaking culture that often culture vessel stirred.
For example, according to the cell culture apparatus described in the patent documentation 1, can make the platform motion of loading culture vessel with various patterns such as rotation, vibrations, thereby can the nutrient solution in the culture vessel be stirred.
In addition, the cell culture apparatus described in patent documentation 2 and the patent documentation 3 adopts following mechanism: the liquid nutrient medium in the culture vessel is vibrated in the mode that does not produce bubble, supply with oxygen so that cell does not produce damage by fluctuation.
By the oscillation method of these cell culture apparatus, whole substratum is stirred tempestuously, so cell separates separately scatteredly, and oxygen and nutritive ingredient are diffused into integral body, can supply to each cell fully.
In addition, in such cell cultures, the propagation of needs cooperation cell maintains the density of the cell in the nutrient solution in the suitable scope.
That is, when the cell density in the nutrient solution is excessive, can not supply with the sufficient oxygen of each cell and nutrition, the cell proliferation decrease in efficiency.In addition, the cell density in the nutrient solution is too small also can not get sufficient proliferate efficiency.
Therefore, when cell cultures, in order in cultivation, to hold cell density, need the cell count in the nutrient solution in the suitable instrumentation culture vessel.
For example, the cell culture apparatus that the propagation that can cooperate cell is suitably kept the cell density in the nutrient solution is disclosed in the patent documentation 4.
When using such cell culture apparatus to carry out the instrumentation of cell count, adopted following method in the past: from culture vessel in the sampling that links to each other with mouthful gather the nutrient solution that contains culturing cell, after the damping fluid of adding regulation is adjusted to the density that is suitable for measuring the cell in the nutrient solution, inject tally, read its quantity by artificial or machinery, thereby calculate cell density.
In addition, the culture apparatus that possesses the shooting unit is disclosed in the patent documentation 5.According to this culture apparatus, can regularly obtain cell image and preservation.
The prior art document
Patent documentation
Patent documentation 1: No. 5057429 communique of United States Patent (USP)
Patent documentation 2: the international brochure that discloses No. 2000/66706
Patent documentation 3: Japanese Unexamined Patent Application Publication 2007-511231 communique
Patent documentation 4: the international brochure that discloses No. 2008/136371
Patent documentation 5: the international brochure that discloses No. 2007/052718
Summary of the invention
But according to the difference of cell category, the propagation of cell is difficult to carry out sometimes under the state of the single dispersion of cell, and when forming the cohesion piece of appropriate size when each cell generation adhesion, proliferate efficiency improves.Particularly, except attached cells such as neural stem cell, embryonic stem cell, liver cell, cornea stem cell, islet cells, can also enumerate suspension cells such as hemocyte.
Under the situation of such cell, leaving standstill in the cultivation in the past, stir culture container tempestuously when at every turn shifting, therefore, the single dispersion of cell this moment, the problem that exists the proliferate efficiency of so-called cell to descend.
In addition, utilizing cell culture apparatus described in the patent documentation 1～3 etc. to carry out under the situation of shaking culture, too substratum integral body is carried out violent stirring, become the state of cell dispersion suspension in substratum, therefore, cell is difficult to form the cohesion piece of appropriate size, has the optimized problem of proliferate efficiency that is difficult to make cell.
In addition, using the cell culture apparatus described in the patent documentation 4 to count under the situation of the cell in the nutrient solution, need in culture vessel, gather nutrient solution, thereby make culture system open, therefore, have the risk of sneaking into pollutions such as assorted bacterium.
In addition, according to the culture apparatus described in the patent documentation 5, though can obtain cell image, be difficult to according to this image instrumentation cell count and obtain cell density exactly.
That is, when utilizing the shooting unit described in the patent documentation 5 that the cell in the culture vessel is taken, by the cell count of instrumentation cell image and with the volume of this cell count divided by the nutrient solution in the visual field of this shooting unit, can calculate cell density.
But, under the situation of the cell in such direct viewing cell container, as shown in figure 24, when the big and cell overlap of the density of cell, instrumentation cell count exactly.In addition, when the density of cell is too small, be difficult to the density of predicted entire, have the problem of the precision reduction of the cell density that calculates.
Using like this under the situation of taking the cell in the direct instrumentation culture vessel in unit, compare with the situation of using existing tally to carry out actual measurement, the thickness of tally is generally about 0.1mm, and the thickness of culture vessel is about 1～2cm relatively therewith, and its thickness reaches 100～200 times.
Therefore, take the observed cell count in unit certainly, even identical volume density also reaches the firm and hard border of usage count when measuring 100～200 times.Therefore, the heterogeneous mutual respect of cell in the culture vessel is folded, and it is very difficult coming the instrumentation cell count by the cell in the direct observation culture vessel.
Therefore, the inventor etc. further investigate, the result, regulate the thickness of culture vessel, make from taking after but the observed cell count in unit reaches the quantity of instrumentation, come cell count in the instrumentation culture vessel by direct observation, successfully obtained the cell density that approaches with measured value thus.
The present invention is the invention of In view of the foregoing finishing, its purpose is, the decomposition control of formation control by carrying out the cell condensation piece in the culture vessel, cohesion piece is provided and will condenses piece and be adjusted to suitable size, thereby can improve cell culture apparatus and the cell culture processes of the proliferate efficiency of cell.
In addition, the objective of the invention is to, provide and need not the open culture system and not to be subjected under the situation of restriction of proliferating cells density method of counting and the counting device of the counting object in the container of instrumentation cell count in any density range under culture environment.
To achieve these goals, the invention provides a kind of cell culture processes, the use culture vessel carries out, it is characterized in that, by culture vessel is applied external force, carry out at least one side's the control of the decomposition control of the formation control of the cell condensation piece in the culture vessel and cohesion piece, carry out the cultivation of cell thus.
In addition, cell culture apparatus of the present invention, for using the cell culture apparatus of culture vessel culturing cell, it has the loading stage of mounting culture vessel and the agitating member of pressing culture vessel and can moving with the speed along continuous straight runs of regulation with the amount of being pressed into of regulation, agitating member is moved and culture vessel is applied external force, thus the formation of the cell condensation piece in the culture vessel and at least one side of decomposition are controlled.
In addition, the method of counting of the counting object in the container of the present invention, the number that is used for the counting object of the liquid in the instrumentation sealed vessel, wherein, regulate the thickness of at least a portion of container, with at least a portion of the scope that is conditioned as the determination object scope, the number of the counting object in this determination object scope of instrumentation.
In addition, in the method for counting of the counting object in the container of the present invention, the liquid of stirred vessel, make the counting object equalization in the liquid after, the thickness of at least a portion of container is regulated.
In addition, counting device of the present invention, for being used for the number of the counting object of the liquid in the sealed vessel is carried out the counting device of instrumentation, the adjustment means that it has the loading stage of container and at least a portion that comprises the determination object scope of container is adjusted to specific thickness.
In addition, counting of the present invention can also further have following formation with device: possess the agitating member that before the thickness that utilizes adjustment means adjusting container the liquid in the container is stirred.
In addition, counting of the present invention can also further have following formation with device: possess: take the unit, it is taken the counting object in the container; Counting unit, its number to the counting object in the image that photographs is carried out instrumentation; And drive unit, it is the number of counting object not within the limits prescribed the time as instrumentation result of counting unit, drive adjustment means, at least a portion of container is adjusted to the thickness of regulation, so that the number of the counting object in the image reaches in the scope of regulation.
The effect of invention
According to the present invention, when carrying out the cell cultures of cell, tissue, microorganism etc., the cohesion piece can be adjusted to suitable size, thereby can improve the proliferate efficiency of cell.
In addition, according to the present invention, can and not be subjected to instrumentation cell count under the situation of restriction of proliferating cells density in open culture system not.
Description of drawings
Fig. 1 is the figure of formation of the cell culture apparatus of expression first embodiment of the present invention.
Fig. 2 is the figure of formation of the drive unit in the cell culture apparatus of expression first embodiment of the present invention.
Fig. 3 is the summary side elevation of the cell culture apparatus of first embodiment of the present invention.
Fig. 4 is the figure of the decomposition of the formation of the cell condensation piece of expression among the present invention and cell condensation piece.
Fig. 5 is the figure of formation of the cell culture apparatus of expression second embodiment of the present invention.
Fig. 6 is the figure of formation of the cell culture apparatus of expression the 3rd embodiment of the present invention.
Fig. 7 is the figure of principle of the method for counting of the counting object of expression in the container of the present invention.
Fig. 8 is the figure that the counting of expression the 4th embodiment of the present invention is used the formation of device.
Fig. 9 is that expression utilizes the counting of the 4th embodiment of the present invention with the figure of the method for thickness regulation (reducing under the situation of thickness) of the container of device.
Figure 10 is the figure that expression utilizes the counting of the 4th embodiment of the present invention method for thickness regulation of the container of device (increasing under the situation of thickness).
Figure 11 is the figure that the counting of expression the 5th embodiment of the present invention is used the formation of device.
Figure 12 is the figure that the counting of expression the 6th embodiment of the present invention is used the home position of device.
Figure 13 is the figure that the counting of expression the 6th embodiment of the present invention is used the whipped state of device.
Figure 14 is the counting of expression the 6th embodiment of the present invention regulated and waited for the state (reducing under the situation of thickness) of precipitation with the thickness of device figure.
Figure 15 is the figure that the counting of expression the 6th embodiment of the present invention is used the microscopic examination state of device.
Figure 16 is the counting of expression the 6th embodiment of the present invention regulated and waited for the state (increasing under the situation of thickness) of precipitation with the thickness of device figure.
Figure 17 is the figure that the kind of the agitation condition that the cell culture apparatus of first embodiment of the present invention carries out is used in expression.
Figure 18 is to use the result's of the cell state under each agitation condition that the cell culture apparatus of first embodiment of the present invention carries out conclusion figure.
Figure 19 is the figure of the culture vessel of use in expression embodiments of the invention 1-5 and the comparative example 1.
Figure 20 is the figure of the image of the cell in expression embodiments of the invention 1 and the comparative example 1.
Figure 21 is the result's of expression embodiments of the invention 1 and comparative example 1 figure.
Figure 22 is the figure of the image of the cell in the expression embodiments of the invention 2～5.
Figure 23 is the result's of expression embodiments of the invention 2～5 figure.
Figure 24 is the figure of the method for counting of the counting object in the existing container of expression.
Embodiment
Below, the preferred implementation to cell culture processes of the present invention and cell culture apparatus describes with reference to the accompanying drawings.
[first embodiment]
At first, with reference to figure 1～Fig. 4 first embodiment of the present invention is described.Fig. 1 is the figure of formation of the cell culture apparatus of expression present embodiment.Fig. 2 is the figure of formation of the drive unit in the cell culture apparatus of expression present embodiment.Fig. 3 is the summary side elevation of the cell culture apparatus of present embodiment.Fig. 4 is the figure of the decomposition of the formation of the cell condensation piece of expression among the present invention and cell condensation piece.
[cell culture apparatus 10]
As shown in Figure 1, the cell culture apparatus 10 of present embodiment has culture vessel 11, loading stage 13, agitating member 14, and enclosing among the resettlement section 11-1 of culture vessel 11 has nutrient solution (substratum) and cell, and is connected with pipe 12.
In addition, culture vessel 11 has the required ventilation property of cell cultures, and in order to confirm content, part or all has the transparency.As the material of the culture vessel that satisfies such condition, for example can enumerate: polyolefine, ethylene-vinyl acetate copolymer, styrenic elastomerics, polyester thermoplastic elastomer, silicone thermoplastic elastomer, silicone rubber etc.
As the material of this pipe 12, suitably select to get final product according to environment for use.For example can use silicone rubber, the flexible vinyl chloride resin, polybutadiene, ethylene-vinyl acetate copolymer, polyvinyl chloride (PVC) RESINS, the polyurethanes thermoplastic elastomer, the polyester thermoplastic elastomer, the silicone thermoplastic elastomer, the styrenic elastomerics, SBS (styrene-butadiene-styrene) for example, SIS (styrene-isoprene-phenylethene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resin, fluorine resin etc.
This above loading stage 13 in, erect at four jiaos of places of the part of mounting culture vessel 11 and to be provided with stationary member 13-1.On the other hand, at four jiaos of culture vessel 11, be equipped with embed (Department and go into) the hole 11-2 of stationary member 13-1.
By each stationary member 13-1 is passed through in each hole 11-2 of culture vessel 11, culture vessel 11 can be fixedly installed on loading stage 13 above.In addition, can prevent that culture vessel 11 is offset along with the movement of agitating member 14.
Need to prove that stationary member needn't be defined in above-mentioned member, then can use various members as long as have the mechanism that can prevent culture vessel 11 skews.
Agitating member 14 is controlled the formation of the cell condensation piece in the culture vessel 11 and the decomposition of cell condensation piece by culture vessel 11 being applied external force.
In the cell culture processes of present embodiment, as shown in Figure 3, when utilizing agitating member 14 to press culture vessel 11 with the amount of being pressed into of regulation, make this agitating member 14 mobile abreast with speed and the loading stage 13 of regulation.This circulation of moving to stipulate repeats to carry out.As this agitating member 14, for example can use roller.
Like this, according to present embodiment, apply external force by using 14 pairs of culture vessels of agitating member, can carry out fine adjustment to the stirring in the culture vessel 11, thereby can be suitable for the stirring of cell condensation piece formation and be suitable for the stirring that the cell condensation piece decomposes.
As shown in Figure 1, supporting station 15 is formed by bearing portion and the linking part that links these bearing portions, described bearing portion each positions of loading stage 13 both sides towards above vertically setting, but with free rotation mode the two ends of agitating member 14 are supported.
As shown in Figure 2, this supporting station 15 can move up and down by the bar type electric cylinder 17 (movement in vertical direction action actuator device) that is installed on the connection section below, can be adjusted in the amount of being pressed into of 14 pairs of culture vessels 11 of agitating member of installing on the supporting station 15 with 0.1 millimeter level subtly.
In addition, bar type electric cylinder 17 is installed on the transfer table 16 on the sliding-type electric cylinder 21 (horizontal motion action actuator device), moves with respect to loading stage 13 along continuous straight runs.In addition, be adjusted in the translational speed of the agitating member of installing on the supporting station 15 14 by the translational speed of along continuous straight runs of control transfer table 16.
Drive unit in the cell culture apparatus 10 of present embodiment has supporting station 15, transfer table 16, bar type electric cylinder 17, sliding-type electric cylinder 21 etc.
Like this, cell culture apparatus 10 according to present embodiment, regulate the amount of being pressed into of 14 pairs of culture vessels 11 of agitating member, the translational speed of agitating member 14 by using bar type electric cylinder 17 and sliding-type electric cylinder 21, can control the stirring of the nutrient solution in the culture vessel 11 thus, thus can be with the size adjustment of cell condensation piece to optimum size.
Need to prove, operation control for agitating member, except using the such electric actuator of bar type electric cylinder 17, sliding-type electric cylinder 21, can also use the actuator that utilizes air pressure, oil pressure, electromagnetic force or adopt the formation of using electric motor, cam.
The formation of＜cell condensation piece 〉
Here, as shown in Figure 4, during cell cultures, according to the difference of cell category, there is the size that is suitable for cultured cells cohesion piece.
That is, culturing cell has following character: the split speed of individual cells is low, and adhesion just begins sufficient division after forming certain cohesion piece mutually.
Common leaving standstill in the cultivation, under the low state of the cell density at the initial stage of cultivation, cell adheres to by diffusion and forms the cohesion piece gradually, but its speed is slow.
Therefore, in the past at the cultivation initial stage, for example use microwell plate that cell is assembled to high-density at a position and make cell be easy to adhere to, perhaps use the container of low capacity and use bigger container along with the propagation of cell, cultivate when cell density from descending preventing thus.
Relative therewith, in the present embodiment, make the cell that floats on the bottom surface in the culture vessel 11 mobile energetically by stirring, improve the probability that adhesion takes place between the cell thus, thereby can form the cohesion piece of appropriate size quickly.
Thus, according to the cell culture processes of the cell culture apparatus 10 that uses present embodiment, can be when cell cultures begins, when the state of the single dispersion of cell, make cells contacting and promote the formation of the cohesion piece of appropriate size, thereby can improve cell proliferation efficient.
In addition, the initial stage (during inoculation) that the formation control of such cell condensation piece is not limited to cultivate, for example in the cultivation of cell, culture vessel 11 is applied situation inferior also being fit to that excessive external force, result causes condensing piece disintegration, the single dispersion of cell and carry out, can improve cell proliferation efficient.
The decomposition of＜cell condensation piece 〉
On the other hand, when the cell condensation piece is excessive, oxygen and the under-nutrition of cohesion piece inside, proliferate efficiency descends.
Therefore, when cohesion piece gigantism, preferably in nutrient solution, produce high current moving (turbulent flow) so that the cohesion piece decomposes.
Cultural method according to the cell culture apparatus 10 that uses present embodiment, when cohesion piece gigantism, by regulating the amount of being pressed into and the translational speed of agitating member 14, control stirring so that produce the moving mode of high current in the nutrient solution, can make the cohesion piece be decomposed into suitable size.
As mentioned above, cell culture apparatus 10 and the cell culture processes that has used this device according to present embodiment, can be when utilizing agitating member 14 to press culture vessel 11 with the amount of being pressed into of regulation, make this agitating member 14 mobile abreast with speed and the loading stage 13 of regulation, thereby can suitably control the intensity that is applied to the external force on the culture vessel.
Therefore, thereby can carry out fine adjustment to the stirring in the culture vessel 11 and be suitable for the stirring that the cell condensation piece forms, and the cohesion piece is decomposed, thereby the cell condensation piece can be adjusted to the size that is suitable for breeding.
[second embodiment]
Below, describe with reference to 5 pairs of second embodiments of the present invention of figure.This Fig. 5 is the figure of formation of the cell culture apparatus of expression present embodiment.
Present embodiment is different with first embodiment in the following areas: using partition member culture vessel 11 to be divided into cultivation portion and expansible and can volume of culture being adjusted in the cell culture apparatus 10 of suitable size according to the propagation of cell, utilize agitating member (agitating roller) 14 to stir cultivation portion.In addition, in the present embodiment, the two ends of culture vessel 11 are fixing with clip member 23.Other aspects are identical with first embodiment.
Namely, as shown in Figure 5, the cell culture apparatus 10 of present embodiment use to be separated 22 pairs of culture vessels 11 of roller (partition member) and is cut apart, and is provided with the cultivation portion of enclosing nutrient solution and cell and is used for making the expansible portion that roller 22 moves to expand the volume of cultivation portion of separating.This separates roller 22 and agitating member (agitating roller) 14 arranges abreast, and can be mobile abreast with loading stage 13.
By using such separation roller 22, the volume of cultivation portion is changed continuously, separate roller 22 and move and make the volume gain of cultivation portion along with cell proliferation by making, cell density can be maintained in the suitable scope.
In the example of this Fig. 5, adopted and used two to separate rollers 22 and clamp culture vessel 11 and the formation separated from above-below direction, but be not limited thereto, also can adopt by one and separate that roller 22 is pressed culture vessel 11 from the top with respect to loading stage 13 and the formation that culture vessel 11 is divided into cultivation portion and expansible portion.
Need to prove, use such partition member control culture volume to improve the technology of culture efficiency, at length be documented in the world by the applicant's application and disclose No. 2008/136371 brochure and the world and disclose in No. 2008/136339 brochure.
Therefore, can further improve the proliferate efficiency of cell.
[the 3rd embodiment]
Below, describe with reference to 6 pairs of the 3rd embodiments of the present invention of figure.This Fig. 6 is the figure of formation of the cell culture apparatus 10 of expression present embodiment.
Present embodiment is different with first embodiment in the following areas: within the limits prescribed whether the size of taking the cells in the culture vessel 11 and judging the cohesion piece automatically will condense piece based on this result of determination and be adjusted to suitable size.Other aspects are identical with first embodiment.
That is, the cell culture apparatus 10 of present embodiment as shown in Figure 6, except the formation of first embodiment, also has filming apparatus 30 and control device 40.
When filming apparatus 30 receives the indication information of shooting from control device 40, the cell in the culture vessel 11 is taken, and the image of gained is sent to control device 40.Indication information based on the shooting of filming apparatus 30 can be sent by control device 40 automatically on the opportunity of regulation.
As this filming apparatus 30, for example can the CCD camera be installed at the lens barrel of phase contrast microscope and use.
Filming apparatus control part 41 sent for the indication information of taking filming apparatus 30 on the opportunity of regulation, and received the image that photographs from filming apparatus 30.
Whether within the limits prescribed cohesion piece size discrimination portion 42 judges the size of the cell condensation piece in this graphic information when filming apparatus control part 41 receives image.As the scope of this regulation, for example can be set at 100 μ m～600 μ m.As the size of this cohesion piece, can use the mean value of the cohesion piece that photographs etc.
Then, the result of judgement is, the size of cell condensation piece less than or situation greater than specialized range under, this result of determination is outputed to drive control portion 43.
Drive control portion 43 is based on the result of determination from 42 inputs of cohesion piece size discrimination portion, determine the amount of being pressed into, translational speed and the mobile circulation of agitating member 14, according to these drive conditions bar type electric cylinder 17 and sliding-type electric cylinder 21 are controlled.
Thus, under the situation of size less than specialized range of cell condensation piece, make the cell condensation piece in the culture vessel 11 be formed up to suitable size, under the situation of size greater than specialized range of cell condensation piece, make the cell condensation piece decompose to suitable size, can be adjusted to the size that is suitable for breeding most thus.
Need to prove, in order to carry out such processing, preferred control device 40 or drive control portion 43 have the size of storing various cohesion pieces accordingly and are suitable for the amount of being pressed into of the agitating member 14 of each situation, translational speed and the form (table) of mobile circulation etc.
As discussed above, according to cell culture apparatus 10 and the cell culture processes that has used this device of present embodiment, the size that can automatically regulate the cohesion piece of culture vessel 11, thus can stably improve cell proliferation efficient.
Below, describe with reference to the principle of the method for counting of the counting object in 7 pairs of containers of the present invention of figure.
This Fig. 7 is the figure that the state of the interior cell of microscope direct observing culture vessel and its quantity of instrumentation is used in expression, and Fig. 7 (A) expression is such situation of not regulating the thickness of container and observing in the past.Need to prove, by using proportion less than the nutrient solution of cell proportion, can make cell sink to the bottom of container and be suitable for microscopic examination.In addition, on the contrary, also can use heavy nutrient solution that cell aggregation is observed in the top of container.
Under the situation of Fig. 7 (A), along with the increase of the cell count in the container, when after suspending, leaving standstill, can overlap gradually.Therefore, during with a large amount of culturing cell of this method, be difficult to instrumentation cell count exactly.
Therefore, among the present invention,, shown in Fig. 7 (B), by reducing the thickness of container, the cell count in the observation scope (determination object scope) is reduced, thereby be adjusted to the cell count that is suitable for instrumentation too much and accurately under the situation of instrumentation in cell count.
Thus, even under the situation of utilizing a large amount of culturing cells of culture vessel, also can need not the open culture system by the cell in the direct viewing culture vessel and come the instrumentation cell count.
In addition, the cell count in culture vessel is few, be difficult to predict under the situation of density of culture vessel integral body, also can the cell count in the observation scope be increased, thereby be adjusted to the cell count that is suitable for instrumentation by increasing the thickness of container.
[the 4th embodiment]
Below, describe with device with reference to the method for counting of the counting object in the container of 8 pairs of the 4th embodiments of the present invention of figure and counting.This Fig. 8 is the figure that the counting of expression present embodiment is used the formation of device.
The counting of present embodiment device 50, as shown in Figure 8, have: thickness adjustment means 51, loading stage 13, take unit 52, drive unit 53, drive unit 54 and throw light on 55, culture vessel 11 is configured on the loading stage 13, the culturing cell numbers in the culture vessel 11 are carried out instrumentation.
Thickness adjustment means 51 is used for regulating the thickness of the culture vessel 11 on the loading stage 13.In the example of this Fig. 8, thickness adjustment means 51 is made of the pressing plate that has be used to the planar section of pressing culture vessel 11, by press the part of the culture vessel of being made by flexible packing material 11 from the top, can reduce its thickness.
In addition, in the thickness adjustment means 51 at least the upper section of the determination object scope of culture vessel 11 formed by material transparent, 55 illuminate this part from the top by throwing light on, and observe from the below by the shooting unit that constituted by microscope and CCD camera 52.
Need to prove that thickness adjustment means 51 is not limited to the pressing plate that planar section is pressed culture vessel 11 that passes through shown in Figure 8, also can use roller, tractive member and constitute.
For example, utilize roller to clamp culture vessel 11 and movement from up and down two sides or a side, make the horizontal area minimizing of culture vessel 11, can increase the thickness of culture vessel 11 thus.On the contrary, also can reduce the thickness of culture vessel 11 by the horizontal area that increases culture vessel 11.In addition, by using tractive member along continuous straight runs stretching culture vessel 11, also can reduce the thickness of culture vessel 11.
The below part of the determination object scope of this loading stage 13 is made of transparent members such as sheet glass 56, can observe culture vessel 11 from the below by taking unit 52.
This drive unit 53 for example can use bar type electric cylinder (movement in vertical direction action actuator device), thus, can regulate the thickness of culture vessel 11 with 0.01 millimeter level subtly.
In addition, drive unit 54 makes shooting unit 52 move with respect to loading stage 13 along continuous straight runs by ball screw as shown in Figure 8.By this drive unit 54, when the cell in the culture vessel 11 is taken beyond, make and take the outside that unit 52 is disposed at loading stage 13, when taking, can make take unit 52 move to culture vessel 11 the determination object scope below.
Need to prove that drive unit 53, drive unit 54 can use the actuator that utilizes air pressure, oil pressure, electromagnetic force and constitute except electric actuator, perhaps also can use electric motor, cam and constitute.
Below, the example of the counting that utilizes present embodiment being regulated the thickness of culture vessel 11 with device with reference to figure 9 and Figure 10 describes.Fig. 9 represents to reduce the situation of container thickness, and Figure 10 represents to increase the situation of container thickness.
Under the situation of the thickness that reduces culture vessel 11, the thickness of container is regulated except mode shown in Figure 8, for example, shown in Fig. 9 (a), can be undertaken by pressing thickness adjustment means 51 from the below of culture vessel 11.Need to prove that though illustrate, this moment is preferably with the top stationkeeping with culture vessel 11 such as sheet glass.
In addition, but under the situation that culture vessel 11 is made by the material of horizontal stretch, shown in Fig. 9 (b), also can use whole the thickness adjustment means 51 that can cover culture vessel 11 to press the thickness that culture vessel 11 reduces culture vessel 11.In addition, shown in Fig. 9 (c), also can utilize thickness adjustment means 51 stretching culture vessels 11 to reduce its thickness.In this case, can adopt the constituting of the fixing edge that utilizes the thickness adjustment means 51 tractive horizontal direction opposition sides that constituted by the tractive member simultaneously of an end at the edge of culture vessel 11, perhaps also can adopt two of horizontal direction by using two thickness adjustment means 51 tractive culture vessels 11 to bring in the formation of stretching culture vessel 11.Material as the culture vessel 11 of such softness for example can use silicone rubber etc.
In addition, shown in Fig. 9 (d), also can use roller etc. as thickness adjustment means 51, by moving it, the horizontal area of culture vessel 11 be increased, thereby reduce the thickness of culture vessel 11.
On the other hand, under the situation of the thickness that increases culture vessel 11, for example, shown in Figure 10 (e), can utilize thickness adjustment means 51 to press the top part of culture vessel 11, the thickness of the part part in addition that is pressed of culture vessel 11 is increased.
In addition, shown in Figure 10 (f), also can use roller etc. as thickness adjustment means 51, by moving it, the horizontal area of culture vessel 11 be reduced, thereby increase the thickness of culture vessel 11.
Below, illustrate in greater detail the method for counting of the counting object in the container of present embodiment with reference to figure 8.
At first, before the step of the cell count in instrumentation culture vessel 11, preferably the nutrient solution in the culture vessel 11 is stirred.Need to prove, about the agitating unit of nutrient solution, in the 5th embodiment, describe in detail.
Then, use drive unit 54, make and take the below that unit 52 moves to the determination object scope of culture vessel 11.
Then, use drive unit 53, thickness adjustment means 51 is descended, the thickness of culture vessel 11 is adjusted to the thickness of regulation.
At this moment, the area of the size of the kind that the thickness of regulation can be by cell, culture vessel, determination object scope and incubation time etc. obtain various values.
Then, utilize illumination 55 to illuminate the determination object scope, and utilize shooting unit 52 to take.Then, the cell on the image that photographs of counting.At this moment, send the image that photographs from taking unit 52 to counting unit, by this counting unit instrumentation cell count automatically.As such counting unit, can use known cell scaler analyzer, cell count measuring device etc.
Here, in the present embodiment, by using proportion less than the nutrient solution of culturing cell, make culturing cell be deposited to the bottom of culture vessel 11, can take them by making the cell of precipitation in focus of taking the unit.But, under the situation of the cell overlap in, the determination object scope big at cell density, counting cells number exactly.
Therefore, in the method for counting of the counting object in the container of present embodiment, under the situation of the cell overlap in determination object scope like this, by thickness adjustment means 51 being moved reduce the thickness of culture vessel 11, thereby reduce the cell count of instrumentation object, can the instrumentation cell.
Need to prove, can think not overlapping and maximum cell number that can observe is about the number that obtains divided by the mean level (ML) area of culturing cell less than the area with the determination object scope in the determination object scope.Therefore, be that cell count is calculated as under this situation more than maximum cell number in Cytometric result, the thickness that preferably further reduces culture vessel 11 is counted again.In addition, also can infer the quantity when cell is closely arranged more accurately in the determination object scope, use this presumed value as the maximum cell number etc., other values are used for judging whether to carry out the thickness adjusting of culture vessel 11.
In addition, be under the situation of cell count less than certain value in Cytometric result, because the precision of the cell density of gained reduces, therefore, count again after preferably increasing the thickness of culture vessel 11.Instrumentation device about such thickness that makes culture vessel 11 increases describes in detail in the 6th embodiment.
So during the cell count in the instrumentation determination object scope, by with the volume of this cell count divided by the determination object scope, can calculate cell density.And then, multiply by the volume of culture vessel 11 by the cell density with gained, can calculate the cell count of culture vessel 11 integral body.
The cell count of such cell density and culture vessel 11 integral body also can automatically be calculated by counting unit.
Like this, according to present embodiment, even the cell count in culture vessel 11 is many, when directly observing culture vessel 11 exactly under the situation of instrumentation cell count, also can come the instrumentation cell count by the thickness that reduces culture vessel, thereby can calculate the cell density in the culture vessel 11.
[the 5th embodiment]
Below, with reference to Figure 11 and Fig. 2 the method for counting of the counting object in the container of the 5th embodiment of the present invention and counting are described with device.Figure 11 is the figure that the counting of expression present embodiment is used the formation of device.Fig. 2 is the figure of the formation of the drive unit (agitating member with) in the cell culture apparatus of expression first embodiment, also can use same drive unit in the present embodiment.
The counting device of present embodiment except the counting of the 4th embodiment is used the formation of device, also possesses agitating member.In the present embodiment, by using the nutrient solution in this agitating member stir culture container 11, cell is disperseed in the mode that is easy to observe.Other aspects can be same with the 4th embodiment.
Agitating member 14 stirs the nutrient solution in the culture vessel 11 by moving when pressing to culture vessel 11, thereby the culturing cell in the nutrient solution is disperseed.As this agitating member 14, for example can use roller as shown in figure 11.
In the example of this Figure 11, adopt following formation: press culture vessel 11 by utilizing agitating member 14 with the amount of being pressed into of regulation, this agitating member 14 is moved abreast repeatedly with the speed of regulation and circulation and the loading stage 13 of regulation, nutrient solution is stirred.
As shown in figure 11, but supporting station 15 by each positions of loading stage 13 both sides towards above the bearing portion at two ends of the free rotary ground supporting agitating member 14 that vertically arranges and the linking part that links these bearing portions form.
In addition, as shown in Figure 2, this supporting station 15 can move up and down by the bar type electric cylinder 17 (movement in vertical direction action actuator device) that is installed on the connection section below, can regulate the amount of being pressed into of the 14 pairs of culture vessels 11 of agitating member that are installed on the supporting station 15 with 0.1 millimeter level subtly.
In addition, bar type electric cylinder 17 is installed on the transfer table 16 on the sliding-type electric cylinder 21 (horizontal motion action actuator device), moves with respect to loading stage 13 along continuous straight runs.In addition, the translational speed of regulating the agitating member 14 that is installed on the supporting station 15 by the translational speed of along continuous straight runs of control transfer table 16.
Need to prove, operation control for agitating member 14, except using the such electric actuator of bar type electric cylinder 17, sliding-type electric cylinder 21, can also use the actuator that utilizes air pressure, oil pressure, electromagnetic force or adopt the formation of using electric motor, cam.
Method of counting and counting device according to the counting object in the container of present embodiment, before can the cell count in instrumentation culture vessel 11, pressing under the state of agitating member 14 to culture vessel 11 with the amount of being pressed into of regulation, make agitating member 14 along continuous straight runs carry out the back and forth movement of certain hour.
Thus, can the nutrient solution in the culture vessel 11 be stirred, thereby the culturing cell in the culture vessel 11 is disperseed in the mode that is easy to count.
[the 6th embodiment]
Below, with reference to Figure 12～Figure 16 the method for counting of the counting object in the container of the 6th embodiment of the present invention and counting are described with device.These figure be represent respectively in the method for counting of the counting object in the container of present embodiment counting with home position (Figure 12), whipped state (Figure 13), the thickness of device regulate and wait for precipitation state (will reduce under the situation of the cell of counting, Figure 14), microscopic examination state (Figure 15), thickness is regulated and wait for the synoptic diagram of the state that precipitates (will increase under the situation of the cell of counting, Figure 16).
The counting device of present embodiment is except the counting of the 5th embodiment is used the formation of device.Also possesses the thickness adjustment means that increases be used to the thickness that makes culture vessel 11.Other aspects can be same with the 5th embodiment.
Below, with the working order of device, the operation that the operation that the thickness that makes culture vessel 11 is increased and the thickness that makes culture vessel 11 reduce is included to be described for the counting in the method for counting of the counting object in the container of present embodiment.
(1) home position
At first, describe with reference to the counting of the present embodiment of Figure 12 home position with the integrant of device.
Among this Figure 12, be provided with the spy hole of observation by microscope at loading stage 13, the top of this spy hole is inlaid with the sheet glass 56 of a top part that constitutes loading stage 13.
In addition, an end at the edge of culture vessel 11 disposes thickness adjustment means 51-2 (roller), by moving it, can reduce the horizontal area of culture vessel 11, thereby increases thickness.
In addition, the top of culture vessel 11 disposes agitating member 14 (roller), by make this agitating member 14 downwards under the mobile state that is pressed on the culture vessel 11 along continuous straight runs move, can stir the nutrient solutions in the culture vessel 11.
In addition, when home position, the outside of loading stage 13 disposes microscope 52-1 and the CCD camera 52-2 of the shooting unit 52 that is configured for taking cell and throws light on 55.
(2) whipped state
Below, describe with reference to the order that before the counting of Figure 13 for the number of the counting object in carrying out container the nutrient solution in the culture vessel 11 is stirred.
At first, make agitating member 14 mobile from the home position of Figure 12 downwards, press culture vessel 11 with the amount of being pressed into of regulation.Then, this agitating member 14 is moved abreast repeatedly with the speed of regulation and circulation and the loading stage 13 of regulation.
Thus, can stir the nutrient solutions in the culture vessel 11, thereby make cell equalization in the nutrient solution.
(3) state (reducing under the situation of thickness) of precipitation is regulated and waited for to thickness
Below, thereby reduce to make the hypocellular order of counting to describe with reference to Figure 14 for the thickness that makes culture vessel 11.
At first, agitating member 14 is moved upward and turns back to the home position of Figure 12, and make thickness adjustment means 51-1 mobile downwards, the thickness of culture vessel is reduced.At this moment, by thickness adjustment means 51-1 the part of the culture vessel 11 in the zone on the spy hole that comprises loading stage 13 is pressed, the thickness of the determination object scope of culture vessel 11 is adjusted to the size of regulation.At this moment, under culture environment, the thickness of culture vessel 11 is more little, more can reduce the cell of counting.
After the thickness of culture vessel 11 was reduced, the cells that leave standstill to the culture vessel 11 precipitated.
(4) microscopic examination state
Below, describe carrying out Cytometric order with reference to Figure 15.
Cell under the state that the thickness that makes culture vessel 11 reduces in the culture vessel 11 has carried out post precipitation, as shown in figure 15, make the shooting unit 52 that is constituted by microscope 52-1 and CCD camera 52-2 and the 55 home position along continuous straight runs from Figure 12 that throw light on move, make take unit 52 be disposed at spy hole under, make illumination 55 be disposed at the top of spy hole.Then, take by the cell of precipitation in the culture vessel 11 of CCD camera 52-2.
The image that obtains like this is input in the not shown counting mechanism, by the cell count in this counting device determining image, with the cell count of the gained volume divided by determination object scope (zone of the culture vessel 11 that is observed by CCD camera 52-2), can calculate the cell density in the culture vessel 11 thus.
Thus, even the cell proliferation in culture vessel 11, when under original thickness, precipitating, under the situation that overlaps and can't count exactly, also can count by the thickness of regulating culture vessel 11, thereby can under the situation that need not the open culture system, calculate cell density in the culture vessel 11.
(5) state (under the situation that increases thickness) of precipitation is regulated and waited for to thickness
On the other hand, in the initial stage of cultivating etc., because cell count is few, though therefore can count, the precision of the cell density of gained reduces sometimes.
Therefore, under the few situation of the cell count that observes, as shown in figure 16, the thickness of culture vessel 11 is increased, cell count in the determination object scope is increased after, carry out microscopic examination of explanation in (4).
Namely, after the stirring of (2), agitating member 14 is moved upward and turns back to the home position of Figure 12, make the thickness adjustment means 51-2 that is constituted by roller move and culture vessel 11 is pressed along determination object scope direction, the thickness of culture vessel 11 is increased.At this moment, for the thickness that makes the determination object scope in the culture vessel 11 keeps certain, make thickness adjustment means 51-1 move on the determination object scope top position contacting with culture vessel 11.Then, the cells that leave standstill to the culture vessel 11 precipitate.
Thus, the cell count in the determination object scope is increased, under the few situation of the cell count in culture vessel 11, can calculate cell density more accurately.
Cell count concrete cell count after a little while in the culture vessel 11 suitably determines according to the kind of cell, the area of determination object scope etc., for example can be set at 0 or less than numbers such as 10.
Need to prove, according to the sequential adjustment of above-mentioned (1)～(5) behind the thickness of culture vessel 11, in the determination object scope, observe the overlapping of cell or do not observe cell or almost do not observe under the situation of cell, according to the thickness of the sequential adjustment culture vessel 11 of (1)～(5), carry out the counting of cell count more again.
Thus, according to present embodiment, cell count in culture vessel 11 is few, may can not get when directly observing culture vessel 11 increasing by the thickness that makes culture vessel under the situation of cell density accurately, can calculate the cell density in the culture vessel 11 more accurately.
Embodiment
Below, the embodiment that the cell culture apparatus 10 that uses first embodiment is carried out cell cultures describes.At first, with reference to Figure 17 and Figure 18 the degree of the stirring under various culture condition and each situation is described.Figure 17 is the figure that the kind of the agitation condition that the cell culture apparatus 10 of present embodiment carries out is used in expression, and Figure 18 is the result's of the cell state under each agitation condition conclusion figure.
In the cultural method of the present invention, as shown in figure 17, use agitating member 14 to press culture vessel 11 from the top, make this agitating member 14 mobile abreast with respect to loading stage 13, thus the substratum in the culture vessel 11 is stirred.
The amount of being pressed into when this agitating member 14 is pressed to culture vessel 11 can be got various values, but for example as shown in Figure 17, is under the situation of 10.5mm at the thickness of culture vessel 11, can get 2mm, 4mm, 6mm, 8mm equivalence.
Then, the translational speed of regulating agitating member 14 respectively according to the amount of being pressed into respectively is controlled to be the stirring of the formation that is suitable for the cell condensation piece with the stirring of the nutrient solution in the culture vessel 11 thus or is suitable for the stirring of the decomposition of cell condensation piece.
As the translational speed of agitating member 14, for example as shown in Figure 17, can get 2.5mm/ second, 12.5mm/ second, 50mm/ equivalence second.
Need to prove, the diameter of agitating member 14 is not particularly limited, but stir for control exactly that preferably the thickness setting with respect to culture vessel 11 is 0.5 times～3.0 times.
Figure 18 is illustrated in when stirring under the above-mentioned agitation condition, and which kind of degree the nutrient solution in the culture vessel 11 is stirred to respectively.
As shown in Figure 18, the amount of being pressed into be 2mm and translational speed be 2.5mm/ second, 12.5mm/ during second and the amount of being pressed into be that 4mm and translational speed are 2.5mm/ during second, the cells in the culture vessel 11 all are the precipitation state.
In this specification sheets, such stirring is called " weak stirring ".By carrying out the stirring of such " weak stirring ", can promote the formation of cell condensation piece.
In addition, among Figure 18, when the amount of being pressed into be 2mm and translational speed be 50mm/ during second, the amount of being pressed into be 4mm and translational speed be 12.5mm/ second, 50mm/ during second, the amount of being pressed into be 6mm and translational speed be 2.5mm/ second, 12.5mm/ during second and the amount of being pressed into be that 8mm and translational speed are that 2.5mm/ is during second, nutrient solution in the culture vessel 11 is stirred to a certain degree, but cell is almost the precipitation state.In addition, when the amount of being pressed into be 6mm and translational speed be 50mm/ during second and the amount of being pressed into be that 8mm and translational speed are 12.5mm/ during second, the nutrient solutions in the culture vessel 11 are stirred to a certain degree, but cell substantially all floats.
In this specification sheets, such cell is called " middle stirring " for the stirring under the agitation condition of the boundary vicinity of the state of the state of basic precipitation and basic come-up.By carrying out the stirring of such " middle stirring ", can make not excessive decomposition and be adjusted to suitable size of cell condensation piece.
On the other hand, in Figure 18, when the amount of being pressed into is that 8mm and translational speed are 50mm/ during second, the nutrient solutions in the culture vessel 11 are stirred tempestuously, and cell all floats.In this specification sheets, the stirring that such cell is all floated is called " strong mixing ".When like this " strong mixing ", the cell condensation piece is decomposed into single cell dispersedly, and by carrying out " strong mixing " repeatedly, cell becomes suspended state, but cell when becoming the state of single dispersion proliferate efficiency descend on the contrary.
Stirring in the cell cultures is in the past normally carried out violent stirring to culture vessel, and the result is equivalent to carry out " strong mixing ", has the problem that causes proliferate efficiency to descend, but can eliminate such problem by the present invention.
Below, the method for counting by the counting object in the container of the present invention and counting are regulated the embodiment of instrumentation cell count behind the thickness of culture vessel 11 with device and do not regulated the thickness of culture vessel 11 and the comparative example of instrumentation cell count describes.
(embodiment 1)
As culture vessel 11, used the bag of LDPE (linear low density polyethylene) system, thickness 0.15mm.Culture vessel 11 is that 250mm, minor face are 210mm by the long limit of separating, making the zone i.e. cultivation portion of cultivating with partition member (rubber rollers) as shown in figure 19, adds nutrient solution 640ml and use in this cultivation portion.Need to prove that during culture vessel 11 horizontal of this moment, although above the container be not horizontal plane completely, container thickness is about 16mm.The horizontal plane of range of observation (determination object scope) is of a size of the square of length of side 0.5mm.
Use the AlyS505N-0 substratum of cell science institute as nutrient solution, and uses in cell cultures and breed the lymphadenomatous Jurkat E6.1 of human leukemia T strain as aequum in ware (dish) as culturing cell.
Use the roller of diameter 12mm as agitating member 14, in the amount of being pressed into 13mm, speed 50mm/ second, come and go under the agitation condition of number of times 10 times and stir.
After the stirring, make immediately by width 50mm, thickness 3mm, length to descend greater than the thickness adjustment means that the acrylic resin board on the long limit of culture vessel 11 constitutes, the thickness of bag is adjusted to 3.1mm.Then, leave standstill 12 minutes after, observation scope is taken, carry out the counting of cell count.In addition, according to the volume calculation cell density of this cell count and observation scope.The photo of taking is shown in Figure 20, and observation scope inner cell number, cell density, actual density and measured value ratio are shown in Figure 21.
Need to prove that actual density is by utilizing existing method, the cell count in the nutrient solution that usage count plate (OneCell Counter (OneCell corporate system)) instrumentation reclaims from bag obtains divided by the volume of observation scope with this cell count.
(comparative example 1)
Use culture vessel 11 and the nutrient solution identical with embodiment 1, under identical agitation condition, stir.
Then, do not regulate the bag thickness, leave standstill 60 minutes after, observation scope is taken.Because not to pressing above the bag, therefore produce wavy concavo-convexly, the thickness of bag is about 16mm.
In this case, can be observed the overlapping of cell, can't carry out the counting of cell count, also can't carry out the calculating of density.It the results are shown in Figure 20, Figure 21.
(embodiment 2)
Use the different nutrient solution of cell density and embodiment 1, and the thickness setting of bag is 11.0mm, time of repose afterwards is set at 45 minutes during the instrumentation cell count, in addition, experimentize similarly to Example 1.The kind of nutrient solution and culturing cell is identical with embodiment 1, but the nutrient solution that uses the culturing cell number to lack than embodiment 1 in the present embodiment.It the results are shown in Figure 22 and Figure 23.
(embodiment 3)
Except when the instrumentation cell count with the thickness setting of bag be 7.0mm, will after time of repose be set at 30 minutes, experimentize similarly to Example 2.It the results are shown in Figure 22 and Figure 23.
(embodiment 4)
Except when the instrumentation cell count with the thickness setting of bag be 4.0mm, will after time of repose be set at 12 minutes, experimentize similarly to Example 2.It the results are shown in Figure 22 and Figure 23.
(embodiment 5)
Except when the instrumentation cell count with the thickness setting of bag be 3.1mm, will after time of repose be set at 12 minutes, experimentize similarly to Example 2.It the results are shown in Figure 22 and Figure 23.
As Figure 20 and shown in Figure 21, not regulate in the comparative example 1 of bag thickness, cell overlaps, and can't count exactly.Need to prove that can not count can be that in fact imponderable situation and count results are the situation more than the prescribed value (the maximum cell number that can recognize) in observation scope.
Relative therewith, in embodiment 1, by reducing the thickness of bag, can the instrumentation cell count, thus can calculate cell density in the culture vessel 11.
In addition, as Figure 22 and shown in Figure 23, in embodiment 2～5, as can be known along with the reducing of bag thickness, the cell count of institute's instrumentation reduces.The density of the cell that calculates in addition, is the density of comparing no significant difference with actual density.
Therefore as can be known, under many, the incalculable situations of cell count, when cell density is more big, then reduce the thickness of bag more muchly, can carry out instrumentation to cell thus.
By these experimental results as can be known, according to the method for counting that uses counting of the present invention with the counting object in the container of device, can can calculate cell density need not the open culture system and not being subjected to the cell count in the instrumentation culture vessel under the situation of restriction of cell density.
The invention is not restricted to above embodiment, it is self-evident can carrying out various distortion enforcements within the scope of the invention.
For example, can carry out following suitable change: the mode that culture vessel 11 is constituted not produce the angle part makes the angle become the shape of circle or makes agitating member 14 to make cross section shown in Figure 17 be not different shapes such as star into cylindric, to obtain various mixing effects etc.
In addition, in the above-described embodiment and examples,, but be not limited thereto as the instrumentation object with culturing cell, for example also can be with other organisms such as planktonic organism or inorganics as the instrumentation object." liquid " in the culture vessel also comprises semi-fluid except liquid such as nutrient solution.In addition, can use than the heavy nutrient solution of culturing cell as the nutrient solution in the culture vessel 11, can make culturing cell be positioned at the top of culture vessel 11 thus, thereby it is counted.In addition, in the above-described embodiment and examples, not only can counting cells, fertility status that also can observation of cell etc.
Industrial applicability
The present invention can be applicable to fields such as the biological medicine of need cultivating a large amount of cells, regenerative medicine, immunotherapy.
Claims (12)
1. the method for counting of the counting object in the container is used for the number of the counting object of the liquid in the instrumentation sealed vessel, it is characterized in that,
Regulate the thickness of at least a portion of described container, with at least a portion of the scope that is conditioned as the determination object scope, the number of the counting object in this determination object scope of instrumentation.
2. the method for counting of the counting object in the container as claimed in claim 1 is characterized in that,
Liquid in the described container is stirred, make the described counting object equalization in the described liquid after, the thickness of at least a portion of described container is regulated.
3. the method for counting of the counting object in the container as claimed in claim 1 or 2 is characterized in that,
The number of the described counting object that the use instrumentation obtains is calculated the density of the counting object in the described liquid, and/or is calculated the number of the counting object of described liquid integral body.
4. the method for counting of the counting object in the container as claimed in claim 1 or 2 is characterized in that,
The instrumentation of the number of described counting object is undertaken by the number that instrumentation is taken the described counting object in the image that described determination object scope obtains.
5. the method for counting of the counting object in the container as claimed in claim 1 or 2 is characterized in that,
When the number of the described counting object in the described determination object scope is prescribed value when above, the thickness of at least a portion of described container is reduced after, the number of the described counting object of instrumentation.
6. the method for counting of the counting object in the container as claimed in claim 5 is characterized in that,
The used thickness adjustment means is pressed described container, and the described container that perhaps stretches reduces the thickness of at least a portion of described container.
7. the method for counting of the counting object in the container as claimed in claim 1 or 2 is characterized in that,
When the not enough prescribed value of the number of the described counting object in the described determination object scope, the thickness of at least a portion of described container is increased after, the number of the described counting object of instrumentation.
8. the method for counting of the counting object in the container as claimed in claim 7 is characterized in that,
The used thickness adjustment means is pressed described container, and the thickness of at least a portion of described container is increased.
9. the method for counting of the counting object in the container as claimed in claim 1 or 2, wherein,
Described container is culture vessel, and described counting object is cell.
10. counting device is used for the number of the counting object of the liquid in the instrumentation sealed vessel, it is characterized in that,
Possess the loading stage that loads described container and the adjustment means that at least a portion that comprises the determination object scope in the described container is adjusted to specific thickness.
11. counting device as claimed in claim 10 is characterized in that,
Possess at the agitating member that utilizes described adjustment means the liquid in the described container to be stirred before regulating the thickness of described container.
12. as claim 10 or 11 described counting devices, it is characterized in that possessing:
Take the unit, it is taken the described counting object in the described container,
Counting unit, its number to the described counting object in the image that photographs carry out instrumentation and
Drive unit, the number that its instrumentation result when described counting unit is described counting object is not within the limits prescribed the time, drive described adjustment means, at least a portion of described container is adjusted to the thickness of regulation, so that the number of the counting object in the described image reaches in the scope of regulation.
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| JP2009-242826 | 2009-10-21 | ||
| JP2009242826A JP5659479B2 (en) | 2009-10-21 | 2009-10-21 | Cell counting method and cell counting apparatus in culture vessel |
| CN201080011026.6A CN102348794B (en) | 2009-03-09 | 2010-03-02 | Cell culture method, cell culture device, method for counting subject matters to be counted in container and device for counting |
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| CN201080011026.6A Division CN102348794B (en) | 2009-03-09 | 2010-03-02 | Cell culture method, cell culture device, method for counting subject matters to be counted in container and device for counting |
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| CN103276046B CN103276046B (en) | 2015-07-29 |
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| CN201310220585.3A Active CN103276046B (en) | 2009-03-09 | 2010-03-02 | The method of counting of the count target thing in container and device for counting |
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| KR20140071440A (en) | 2014-06-11 |
| KR20150043561A (en) | 2015-04-22 |
| US9388376B2 (en) | 2016-07-12 |
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| CN103276046B (en) | 2015-07-29 |
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| EP2772533B1 (en) | 2017-02-01 |
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| US20110318725A1 (en) | 2011-12-29 |
| KR101828916B1 (en) | 2018-03-29 |
| KR20130031897A (en) | 2013-03-29 |
| CN102348794B (en) | 2014-09-03 |
| EP2407533A4 (en) | 2013-09-25 |
| EP2407533B1 (en) | 2016-06-15 |
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